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Abstract

Objective— Elevated iron stores and high plasma iron concentration have been linked to an increased risk of atherosclerosis. Iron may thereby affect the interaction of monocytes to endothelium, an initial event in the formation of atherosclerotic plaques.

Methods and Results— Addition of 10 μmol/L non–transferrin-bound iron to the incubation medium caused a 2-fold increase in monocyte adhesion to human umbilical vein endothelial cells (HUVECs). A concordant increase in the expression of the following adhesion molecules was observed: vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and endothelial selectin on HUVECs as well as very late antigen-4, and lymphocyte function–associated antigen-1 on monocytes. The inclusion of either deferiprone or salicylaldehyde isonicotinoylhydrazone counteracted these effects. Intracellular iron chelation by deferoxamine was completed only after 10 hours of incubation, shown by reversal of iron-quenched intracellular calcein signal, and concurrently the effects of iron were blunted. The membrane-impermeable chelator, diethylenetriamine pentaaceticacid, failed to negate iron effects, even after 48 hours of treatment. Furthermore, only membrane-permeable superoxide or hydroxyl radical scavengers were capable of preventing HUVEC activation by iron.

Conclusions— Non–transferrin-bound iron increases the level of intracellular labile iron, which promotes monocyte recruitment to endothelium and may thereby contribute to the pathogenesis of atherosclerosis. Iron-induced adhesion molecule expression was observed, and this event may involve the production of oxygen radicals.

Atherosclerosis has been associated with several important environmental and genetic risk factors. It is characterized by inflammatory changes leading to plaque formation and, furthermore, to plaque rupture and arterial thrombosis. Transendothelial migration of leukocytes is a fundamental inflammatory mechanism in atherogenesis.1 This process is partly mediated by the interaction between endothelial adhesion molecules and their ligands on monocytes.

Elevated concentrations of adhesion molecules have been observed in human atherosclerotic plaques, including 2 members of the immunoglobulin superfamily of adhesion receptors, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as a member of the selectin family, endothelial selectin (E-Selectin). Moreover, a significant correlation has been found between the degree of macrophage infiltration and endothelial ICAM-1, VCAM-1, and E-selectin expression in atherosclerotic lesions.2

The infiltration of leukocytes consists of consecutive adhesion-mediated events. The first step of adhesion involves binding of selectins to carbohydrate ligands that triggers tethering of the leukocytes to the activated endothelium along the vessel wall. Arrest and firm adhesion of the leukocytes on activated endothelial cells occur depending on the activation of the integrins very late antigen-4 (VLA-4) and lymphocyte function–associated antigen-1 (LFA-1).3

In 1981, Sullivan4 suggested that iron depletion may protect against ischemic heart disease. Many epidemiological studies have tested this iron hypothesis. Some showed that the level of body iron stores, as measured by serum ferritin concentration, is positively correlated to the incidence of cardiovascular diseases.5–11 However, others have yielded conflicting results.12–18

Recent studies have demonstrated that carriers of the HFE mutant gene C282Y, responsible for autosomal recessive hereditary hemochromatosis, have a significantly higher plasma concentration of non–transferrin-bound iron (NTBI) than the normal population19 and are at an increased risk of cardiovascular-related death or myocardial infarction.20–22 However, some other studies failed to reveal an increased risk of atherosclerosis in population carrying HFE mutant gene.23–27 A positive association between frequent blood donations and reduced risk of myocardial infarction has been reported,28–30 presumably caused by iron depletion. However, in one study, iron depletion caused by blood donation did not appear to reduce the risk of cardiovascular diseases.31 Additionally, a community-based prospective cohort study showed that heme iron intake was significantly correlated with total iron stores and the risk of cardiovascular diseases.32–35

Given the potential role of iron in atherosclerosis, we investigated the effects of iron-rich and iron-withholding conditions in the course of human monocyte adhesion to human umbilical vein endothelial cells (HUVECs) as one of the earliest events of atherosclerotic plaque formation. Several iron chelators of different cell-membrane permeability were used to investigate the involvement of extracellular and intracellular labile iron in this process. The involvement of toxic oxygen species formed through iron-catalyzed Fenton reaction was also investigated.

Methods

HUVEC Isolation and Culture

HUVECs were isolated and cultured as described by Jaffe et al36 with minor modifications (for details, please see the online Methods, available at http://atvb.ahajournals.org).

Monocyte Isolation

Two different methods of monocyte isolation were used depending on the amount of cells needed (see online Methods): the countercurrent centrifugal elutriation37 and the negative immunoselection monocyte isolation kit (Miltenyi Biotec).

Preparation of Iron Solutions and Chelator-Bound Iron

Two different iron compounds were used in this study. A 10-mmol/L Fe(II)ammoniumsulfate (Merck) solution was made by dissolving the iron crystals in distilled water at room temperature. A 10-mmol/L Fe(III)citrate (Sigma; 1:6 iron-citrate molar ratio) solution was made by dissolving the iron crystals in distilled water at 56°C for 30 minutes. Iron solutions were always freshly prepared and filter-sterilized before use. Fe(III)citrate was used, because the majority of NTBI is found in a complex form of Fe(III) to citrate, as shown by NMR spectroscopy of serum from patients with iron overload.38

The detectable iron from the endothelial basal medium-2, which is the medium used to perform all of the experiments involving HUVECs, was measured by Vitros 950 Chemistry System (Ortho-Clinical Diagnostics; see online Methods). With no external iron addition, the baseline iron level was counted to be ≈0.36 μmol/L. In all experiments, plastic materials with low affinity for iron were used. The contaminant iron level in the working containers and the basal cell culture growth medium is relatively low compared with the iron concentrations used in this study.

In Vitro Cytoadherence Assay

Monocytes and HUVECs were pretreated with different combinations of compounds of interest before the assay. After treatment, monocytes were labeled with 2′,7′-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethylester (BCECF-AM, Molecular Probes). The cytoadherence of monocytes (25×104 cells per well) to HUVEC monolayer (5×104 cells per well) was performed in a 96-well plate for 30 minutes at 37°C with gentle agitation. Fluorescence was read using a filter pair for excitation at 485 nm and emission at 530 nm in a CytoFluor II fluorescence microplate reader (Perseptive Biosystems). This value represented total fluorescing monocytes added to each well. After thorough washing, fluorescence was again measured, and the value represented the remaining monocytes firmly attached to the HUVEC monolayer. The percentage of adhesion was defined as the value of remaining fluorescence divided by the value of total fluorescence multiplied by 100.

Fluorescence-Activated Cell Sorting

After treatment with various compounds of interest, HUVECs or monocytes were incubated with fluorescence-labeled monoclonal antibodies against the adhesion molecules of interest. The level of the adhesion molecule expression on these cells was then analyzed by the FACS analysis using a Becton Dickinson FACScan (see online Methods).

Cellular viability of HUVECs was monitored by the MTT (Sigma) method,40 whereas cellular viability of monocytes after the various compound incubations was monitored using the trypan blue exclusion test (see online Methods).

Calcein-AM Assay

The membrane permeability of iron chelators was followed by the calcein-AM assay41 (see online Methods). One hundred μmol/L of Fe(III)citrate was loaded to confluent HUVECs for 1 hour before the addition of 0.125 μmol/L calcein-AM (30 minutes at 37°C). After the signal was stabilized, the chelator of interest was added. This fluorescence signal (excitation=485 nm; emission=530 nm) was followed for a specified time period. Reversal of the quenched calcein signal by addition of a chelator gives an indication as to the membrane permeability of the chelator at a specific time point.

Data Analysis

Results are expressed as means+SEM. Differences in quantitative measures were tested for significance using the unpaired 2-tailed Student t test.

Results

Cell Viability

After 48 hours of incubation with up to 300 μmol/L Fe(II)ammoniumsulfate or Fe(III)citrate, the viability of HUVECs was >95% as determined by the MTT assay (not shown). TC50 of DF, L1, SIH, and DTPA were also determined. TC50 increased when chelators were bound to iron (Table I, available online at http://atvb.ahajournals.org) indicating their specificity for iron. The MTT assay was also performed to assure that the concentrations of compounds of interest used in all experiments retained 95% cell viability. Monocytes were >95% viable after treatment with <100 μmol/L Fe(II)ammoniumsulfate or Fe(III)citrate for 3 hours shown by the trypan blue exclusion test (not shown).

Iron Modulates Human Monocyte Adhesion to Human Vascular Endothelium

The extent of monocyte adhesion to the endothelium was determined by an in vitro cytoadherence assay. Confluent HUVECs were incubated for 5 hours, whereas monocytes were incubated for 1 hour with Fe(II)ammoniumsulfate. Compared with untreated cells, more iron-treated monocytes adhered to HUVECs and, similarly, more monocytes adhered to iron-treated HUVECs (Figure 1A). A 2-fold increase in adhesion was observed when both endothelial cells and monocytes were pretreated with iron. When HUVECs were incubated with either DF-, L1-, or SIH-saturated iron, this phenomenon was no longer observed (Figure 1B), indicating that the increase in monocyte adhesion to HUVECs was attributed to the presence of iron, the level of which was modulated by external addition of NTBI.

Figure 1. Effects of iron (A) and chelator-bound iron (B) on monocyte adhesion to endothelium (mean±SEM; n=6; *different from control, P<0.05). Source of iron was Fe(II)ammoniumsulfate with incubation time of 5 hours on HUVECs (A and B) and 1 hour on monocytes (A).

Effects of Iron on VCAM-1, ICAM-1, and E-Selectin Expression on HUVECs and the Counter Receptors: VLA-4 and LFA-1 on Monocytes

Incubation of endothelial cells for 48 hours with increasing concentrations of Fe(II)ammoniumsulfate or Fe(III)citrate increased VCAM-1, ICAM-1, and E-selectin expression on HUVECs as observed by the FACS analysis (Figure 2 A through 2D). No difference was seen between Fe(II)ammoniumsulfate and Fe(III)citrate, which may be because of the long incubation time under conditions that promote oxidation of Fe(II). The enhancement of VCAM-1 expression is much more pronounced than ICAM-1 or E-selectin with lower dosage of iron (ie, 1 to 20 μmol/L; Figure 2A). The upregulation of all 3 adhesion molecule expressions was more marked as higher dosage of iron (Figure 2A through 2C) or longer incubation with iron (Figure 3 A and 3B) was applied. These suggest an iron-induced process as dose- and time-dependent, causing an augmentation of HUVEC activation as more iron accumulates intracellularly. Saturating iron with either DF, L1, or SIH completely blunted the effects of iron in VCAM-1 expression (Figure 3C) as well as ICAM-1 and E-selectin (not shown), confirming the involvement of iron in this process.

Figure 3. The expression of adhesion molecules on HUVECs or monocytes after treatment with iron or chelator-bound iron. VCAM-1 expression (mean±SEM; n=3) was lower in 8 hours (A) compared to 48 hours (B) 100 μmol/L Fe(III)citrate-treated HUVEC (P<0.001; n=6). Forty-five hours (B) but not 5 hours (A) of DF treatment was enough to negate iron activation on HUVECs. C, VCAM-1 expression on HUVECs after 48 hours of treatment with iron or chelator-bound iron freshly prepared before being added to the incubation medium (mean±SEM; n=3; *different from control, P<0.001). #Iron was preloaded to HUVECs for 3 hours before a chelator was added for 45 hours. Monocyte surface integrin expression was altered (D) after 1-hour incubation with iron but remained the same as control after 1-hour incubation with DF-bound iron freshly prepared before being added to the incubation medium (mean±SEM; n=3; *different from control, P<0.05).

Subsequent increase in the expression of VLA-4 and LFA-1 was observed on monocytes after 1 hour of 10 μmol/L Fe(III)citrate treatment, suggesting that monocyte activation was attributed to the presence of iron. Incubation with DF-bound iron resulted only in the basal level of expression of these 2 integrins, confirming the involvement of iron in monocyte activation (Figure 3D).

Using the calcein-AM quenching test, the cell-membrane permeability of 4 different chelators was assayed (Figure 4A). L1 and SIH were highly permeant, as after a 2-hour incubation period these 2 chelators could considerably restore the quenched calcein signal. In contrast, DTPA was ineffective at restoring the signal, confirming its membrane impermeability. DF, on the other hand, slowly dequenched calcein signal. Ten hours were needed for 100 μmol/L DF to completely restore the signal previously quenched by 100 μmol/L Fe(III)citrate (Figure 4B). However, <5 hours were required for 10 μmol/L DF to restore the signal quenched by 10 μmol/L Fe(III)citrate.

Figure 4. The calcein-AM assay for assessing the membrane permeability of iron chelators. One hundred percent of fluorescence means no calcein quenching by iron; <100% shows calcein quenching. The time needed for the chelators to enter HUVECs is illustrated as the time to dequench calcein signal after iron treatment (A; mean; n=3). B, Completion of intracellular iron chelation by DF after 10 hours of incubation (mean; n=3).

Treatment with DF for 5 hours on HUVECs pretreated with 100 μmol/L Fe(III)citrate did not fully counteract iron-induced VCAM-1 expression (Figure 3A). After 45 hours of DF treatment (Figure 3B), HUVECs expressed the same amount of VCAM-1 as the non–iron-treated cells. The results suggest that chelation of intracellular iron was necessary to counteract the effects of iron.

A similar experiment was also done using L1 and DTPA (Figure 3C). L1 easily penetrated the endothelial cells and was also capable of counteracting the effects of iron on HUVECs. DTPA, on the other hand, was ineffective. These results confirm the need of chelating intracellular iron to prevent HUVEC activation by iron.

Involvement of Oxygen Radicals in Iron-Promoted Cytoadherence

Promotion of monocyte adhesion to endothelium by iron was no longer observed when HUVECs were coincubated with 5 mmol/L dimethylthiourea (DMTU), a scavenger for both H2O2 and OH. Incubation of confluent HUVECs with H2O2 alone did not induce further monocyte adhesion to endothelium (Figure 5A). These results suggest that iron may produce its effects through the production of reactive oxygen species other than H2O2.

Furthermore, a range of both membrane-permeable and -impermeable scavengers with specific scavenging activity for either O2−, H2O2, or OH· was tested to reveal their involvement in iron-promoted endothelial adhesion molecule expression (Figure 5B). Besides DMTU (H2O2 and OH· scavenger), only thiourea (OH· scavenger) and tempol (O2− scavenger) could counteract the effects of iron on VCAM-1 upregulation. Neither membrane-impermeable scavengers, superoxide dismutase (O2− scavenger), catalase (H2O2 scavenger), and mannitol (OH· scavenger), a membrane-permeable H2O2 scavenger (pyruvate), nor urea (a compound control for DMTU and thiourea) could neutralize the effects of iron. H2O2 alone could not increase the basal expression of VCAM-1. H2O2 in combination with iron gave the same level of VCAM-1 expression as iron alone. Similar results from this set of experiments were observed in endothelial ICAM-1 and E-selectin expression (not shown). These results suggest that iron activates HUVECs through the production of either O2− or OH· or both within the cells.

Discussion

Monocyte migration across the endothelium is an initial pathogenic event of atherosclerosis.1 Iron in vitro has been shown to upregulate interleukin-6 production by HUVECs,42 whereas iron chelators inhibit the tumor necrosis factor-α–mediated upregulation of endothelial adhesion molecules.43,44

In iron overload diseases with 100% transferrin saturation, such as hemochromatosis, a labile form of iron may exist. Furthermore, it is believed that labile iron may also be present in response to other triggers disturbing iron homeostasis. NTBI can be detected in plasma and is bound to ligands with substantially less affinity than transferrin, such as citrate38 and albumin. NTBI is capable of freely entering the cell with no feedback-regulated process.45 Intracellular iron may only be bound to low-molecular weight ligands, catalytically active and capable of initiating free radical reactions.46 The expansion of intracellular labile iron, which may parallel that of NTBI, may lead to cell toxicity.

In this study, we used Fe(II)ammoniumsulfate and Fe(III)citrate as the source of free iron in the incubation medium to investigate the effects of intracellular catalytically-active iron in monocyte interaction to endothelium. Ten μmol/L of iron could directly activate and stimulate the expression of adhesion molecules on both HUVECs and monocytes as well as promote monocyte adhesion to endothelial cells. No enhancement in the adhesion molecule expression and the cytoadherence was observed when chelator-bound iron was added, confirming the involvement of iron in this process.

Proinflammatory cytokines have been shown to be involved in the development of atherosclerosis.1 These cytokines induce iron uptake by monocytes and macrophages.47 Subsequently, our results show an upregulation of monocyte integrin expression due to the presence of labile iron, suggesting that iron may enhance monocyte activation.

L1 and SIH nullified the effect of iron on HUVECs, most probably by chelating intracellular iron. In contrast, DTPA gave no counteracting effect once iron had accumulated. The effectiveness of DF to negate iron effects was time- and concentration-dependent: the higher the level of intracellular iron, the longer the time needed for DF to counteract the effect of iron. This may be because of the route through which DF enters the cells.48 DF enters intact cells by endocytosis. It slowly accumulates in the endosome-lysosome complex, and after longer periods of exposure it is capable of removing intracellular iron. This suggests chelation of intracellular labile iron as an effective way to prevent iron-induced cell activation.

In HUVECs, iron-induced adhesion molecule expression was in a concentration- (0 to 100 μmol/L) and time-dependent manner. Resting levels of iron in the cultured HUVECs might be 10-fold less than in freshly isolated HUVECs,49,50 and ≈1/3 of the amount of NTBI in the loading medium may enter the cells.49 Because NTBI is generally in the range of 1 to 20 μmol/L,51 the results in this study may not only be significant for various conditions of iron overload but also meaningful for normal physiological situations, as an increase in intracellular labile iron in vivo may incline adhesion. This suggests that any condition that disturbs iron homeostasis, even in normal population, may increase the risk for developing atherosclerosis. Furthermore, this study implies that stimulation of adhesion in vivo may reach its lowest point in a state of iron deficiency, which may be protective against atherosclerosis but may also lead to a defective immune function.

Antioxidants have been shown to protect against endothelial dysfunction associated with atherosclerosis,52 whereas iron chelators were capable of preventing hydroxyl radical damage.53 Reactive oxygen species, possibly formed through iron-catalyzed Fenton reaction, may be involved in atherogenesis. In our study, DMTU inhibited iron-induced monocyte adherence to HUVECs. Furthermore, only DMTU and 2 other membrane-permeable scavengers, tempol and thiourea, were able to inhibit iron-induced endothelial adhesion molecule expression, suggesting that O2− and OH· are involved in the process of iron-induced monocyte recruitment to HUVECs.

In conclusion, additional NTBI increases the level of intracellular labile iron. This labile iron may play a role as an independent risk factor in atherosclerosis by promoting monocyte recruitment to endothelium. It modulates the expression of cell adhesion molecules on HUVECs as well as the integrins on monocytes. Iron chelators commonly used for the treatment of secondary iron overload can effectively prevent these iron-mediated events. Furthermore, intracellular labile iron may promote its effect through the production of oxygen radicals, particularly superoxide and hydroxyl radicals.

Acknowledgments

This work was supported by European Commission, QLRT-2001-0044 (http://www.nutrientirontoxicity.nl). We thank J.J.M. Bouwman (Diakonessen Hospital, Utrecht, the Netherlands) for providing the umbilical cords and Prof P. Ponka (McGill University, Montreal, Canada) for providing the iron chelator SIH.